PAR (Protease-Activated Receptor)-2 is a G protein-binding receptor of 7-times transmembrane type found in 1994 by Nystedt et al. (Proc Natl Acad Sci USA, 91, 9208-9212 (1994)). A protease activated receptor (PAR) family in which PAR-1, 2, 3, and 4 have been conventionally known has a unique activation mechanism wherein the activation of PAR is induced by cleaving a specific site in the extracellular N terminal of the receptor molecule, with the action of a protease such as thrombin or trypsin, and then binding a ligand site of the newly exposed cleavage terminal to a binding site of the receptor itself. PAR-1, 3, and 4 are known to be activated by thrombin, while PAR-2 is not activated by thrombin but activated by proteases such as trypsin (Proc Natl Acad Sci USA, 91, 9208-9212 (1994)), tryptase (J Biol Chem., 272(7):4043-4049 (1997)), tissue factor/factor VIIa, factor Xa (Proc Natl Acad Sci USA., 97(10):5255-5260 (2000)), arccosine that is one type of sperm protease (FEBS Lett., 484(3):285-290 (2000)), and trypsin-like serine protease identified from a rat brain (J Neurochem., 74(4):1731-1738 (2000)).
PAR-2 is known to be distributed widely in endothelial tissues, is shown to be expressed at particularly high levels in digestive organs, respiratory organs, blood vessels, skin, kidney and the like, and is suggested to be likely to participate widely in inflammatory diseases because it is activated by trypsin, mast cell-derived tryptase or the like in a living body as described above (Pharmacological Rev, 53, 245-282, (2001)). Actually, pharmacological and genetic analysis in recent years using PAR-2 activating peptides or PAR-2 knockout mice have revealed that the stimulation of PAR-2 exhibits an inflammatory action on many organs (Br J Pharmacol, 125, 419-422 (1998)); the expression of PAR-2 is induced by inflammatory stimulation (J Biol Chem., 271(25):14910-14915, (1996)); PAR-2 is expressed at high levels in inflammatory tissues, atherosclerotic plaques, cancer cells, or the like (J Clin Invest., 111(1):35-41, (2003), Int J Oncol., 23(1):61-66 (2003), or the like); in the PAR-2 knockout mice, a development of inflammations is suppressed in a contact dermatitis model or in an experimental arthritis model (International Publication WO03/049723) and topical infiltration of inflammatory cells causing asthma is suppressed (J Immunol., 165(11):6504-6510 (2000)) or the like, and the action of PAR-2 on inflammations and cancers attracts attention. Accordingly, it is estimated that the prevention of development and progress or amelioration of clinical state for inflammatory diseases (asthma, allergic rhinitis, atopic dermatitis, chronic arthritis and the like) and cancers is made feasible by inhibiting the activation of PAR-2, and the development of PAR-2 activation inhibitors, particularly PAR-2 antagonists, as novel anti-inflammatory agents and anticancer drugs is expected.
In the lacrimal gland and salivary gland, the significant secretion of lacrimal fluid and saliva by PAR-2 activation is recognized, and it is suggested that PAR-2 agonists can serve as therapeutic agents useful for diseases with a reduced secretion problem in the lacrimal gland and salivary gland, such as Sjogren's syndrome (Japanese Patent Application Laid-open Nos. 2001-64203 and 2001-181208). In the digestive organs, there are reports on the protective action, attributable to activation of PAR-2, on gastric mucosa (Japanese Patent Application Laid-open No. 2001-233790), and the promotion, by activation of PAR-2, of autonomic movement of the bowel (U.S. Pat. Nos. 5,888,529 and 5,958,407), and it is estimated that the activation of PAR-2 by PAR-2 agonists would be useful for treatment of gastric ulcer and intestinal obstruction.
As described above, the possibility of PAR-2-targeting agonists or antagonists as therapeutic agents attracts attention, and various methods for evaluation of PAR-2activation have been attempted. For example, the quantification of phosphorylated inositol (Proc Natl Acad Sci USA., 5;94(16):8884-8889 (1997)) or the measurement of intracellular Ca2+ level changes (Anal Biochem., 290(2):378-9 (2001)) has been generally used as a method of using for the production of second messenger accompanying the activation of PAR-2using cells expressing PAR-2. As a method of evaluating the activation ex vivo or in vivo, there are known a method of using the relaxation of an extirpated blood vessel as an indicator (Can J Physiol Pharmacol., 75(7):832-41 (1997)), a method of using salivary hypersecretion as an indicator (Br J Pharmacol., 129(8):1808-14 (2000)), or the like. As a method of directly evaluating the interaction between the ligand and G protein-binding receptor, a receptor-ligand binding test wherein the ligand is labeled with a radioisotope or a fluorescent dye is generally used. There are reports on a receptor-ligand binding test wherein PAR-2-specific ligand trans-cinnamoyl-LIGRLO (SEQ ID NO: 1)-NH2 is used (J Pharmacol Exp Ther, 290, 753-760 (1999)) and on a high-sensitivity assay using highly active PAR-2-activating peptide 2-furoyl-LIGRL (SEQ ID NO: 2)-NH2 (which is being contributed).
However, a report on PAR-2-selective highly active agonists (International Publication WO03/104268) is recognized as described above, but a report on compounds evidently having a PAR-2 antagonist activity is hardly recognized. Up to now, a compound inhibiting an intracellular signal transmission by stimulation with a PAR-2 agonist has been reported (Japanese Patent Application Laid-open No. 2003-286171), however whether the action of the compound is a direct inhibitory action on PAR-2, is not revealed. A series of antagonists described as being derived from PAR-2 agonist structures have been reported (International Publication WO2004/002418), however their inhibitory activity cannot be said to be satisfactory because the mechanism of PAR-2 inhibition is not revealed and further because the concentration thereof for inhibiting PAR-2 stimulation is shown to be in the order of mM. In addition to those described above, peptides derived from PAR-1 or PAR-2 activating peptides reported by Al-Ani et al. are reported to suppress the activation of PAR-2 by stimulation with trypsin, but do not exhibit an inhibitory effect on PAR-2 activating peptides, and are suggested to inhibit the binding or interaction between trypsin and PAR-2. As another unique method of inhibiting PAR-2 activation, there is an approach of specifically inhibiting the signal transmission by preventing the receptor from binding to G protein by using a compound (Pepducin) having palmitic acid added to a peptide mimicking an intracellular domain structure of PAR-2 receptor (Nat Med. 2002 October; 8(10):1161-5), but a use of this approach as pharmacotherapy still has a problem with respect to suitable delivery of the compound to a target site, specificity of receptor signal or the like.
Accordingly, the object of the present invention is to provide a PAR-2 antagonist acting competitively on a ligand-binding site of the receptor by inhibiting the activation of the PAR-2 accurately at the receptor level. That is, the object of the present invention is to provide a pharmaceutical preparation useful for prevention of development and progress, amelioration of clinical state, treatment or the like for PAR-2-associated diseases, for example, respiratory diseases such as asthma, allergic diseases such as allergic rhinitis, cardiovascular system diseases such as myocardial infarction, nervous system diseases such as neuralgia, inflammatory diseases such as atopic dermatitis and chronic arthritis, and cancers.